GSM6620452: wild-type mTEC at air/liquid interface for 3 days, replicate 1; Mus musculus; RNA-Seq
To generate single-cell suspensions for scRNA-seq from mTEC cultures, mTECs derived from wild-type mice and Foxj1-CreERT Rosa-tdTomato mice treated with 4-OHT on day two at air/liquid interface were cultured and differentiated. On day three at air/liquid interface, mTECs were washed with PBS three times both apically and basally. Trypsin-EDTA at a concentration of 0.05% in Cell Dissociation Buffer (Gibco, Cat #13151014) was added both apically and basally and cells were incubated at 37°C for 10 min. Cells were lifted from the membrane with gentle pipetting and trypsin deactivated in mTEC Basic with 2% Nu-Serum (Corning, Cat #355100). Fresh 0.05% Trypsin-EDTA in Cell Dissociation Buffer was added to the apical surface and incubated at 37°C for 5 min. The pipetting, inactivation, and 5 min incubation steps were repeated twice. Cells were washed in FACS buffer (2% FBS, 2% BSA, 5 mM EDTA, 25 mM HEPES in PBS) filtered through a 100 uM cell strainer and resuspended in FACS buffer with Fixable Viability dye (Invitrogen, Cat #65-0863-14) at a dilution of 1:1600. After a 15 min incubation on ice, cells were washed with FACS buffer and sorted on a fluorescence activated cell sorter (Sony SH800). tdTomato-expressing Foxj1-CreERT Rosa-tdTomato cells, representing cells within the multiciliated cell lineage, were sorted into mTEC Basic with 2% Nu-Serum. Wild-type cells and a fraction of Foxj1-CreERT Rosa-tdTomato cells were not sorted. Both tdTomato-expressing and unsorted cells were resuspended in 0.04% BSA in PBS at 1000 cells per uL to prepare for scRNA-seq. Library was performed according to the manufacter's instructions (Chromium Next GEM Single Cell 3' Reagents kit, version 3, 10x Genomics). Briefly, mTECs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.